View source for Glucose Uptake Assay

==Materials==
*2-Deoxyglucose (cold): Sigma D-6134.  Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]
*0.5% BSA in [[KRBH Buffer]]
*Radioactive <sup>14</sup>C-2-deoxyglucose
*Hot 2-DG.  Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG

==Protocol==
#Starve cells >3h in 0.5% FBS
#Prepare insulin in KRBH/BSA (0.6uL insulin/mL, needing 0.5 mL per well)
#Wash cells 2x with warm PBS -/-
#Add 450 uL KRBH/BSA with or without insulin to wells.  Typically do triplicate measurements
#Wait 30 min
#Add 50 uL hot 2-DG solution per well and start timer
#After 5 min add 50 uL cold 2-DG
#Wash cells 3x1mL with cold PBS
#Add 500 uL PBS per well
#Scrape cells with an upside down p200 tip
#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
#Do a bradford assay on 20 uL of cells (use PBS as blank)

==Analysis==
*For excel fill in orange cells in this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/Excel%20Templates/2-DG%20Template.xlsx]
*For sweave use this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/R/Sweave/2-dg%20analysis.Rnw]

[[Category: Metabolic Measurements]]
[[Category: Glucose Uptake]]
[[Category: Cell Culture]]